Most of the techniques for determining analyte(s) have gradually evolved into devices which are increasingly simple to use, allowing the development of rapid and low cost routine diagnostic methods.
This change was particularly significant in the medical field, with the emergence of the diagnostic by “point of care” or “home testing”, in which a diagnostic is directly carried out next the patient bed or at home, without the need for using analytical laboratory automated techniques.
Immunoassays are Part of the Technologies Used for this Type of Rapid Diagnostic.
These immunoassays encompass devices and methods of diagnostic based on affinity binding reactions between specific binding pair members.
Globally, these immunoassays are divided into two well known main approaches.
In the so-called “competition” approach, the desired analyte and a labeled detection reagent are in competition for specifically binding to a capture reagent. The presence or the absence of the desired analyte in the sample is measured, respectively, by the absence or the presence of a visible (or measurable) signal at the capture reagent.
In the so-called “sandwich” approach, a labeled detection reagent binds to the desired analyte, the latter being immobilized on the solid support via the capture reagent. The presence or the absence of the analyte in the liquid sample is measured, respectively, by the presence or the absence of a visible (or measurable) signal at the capture reagent.
Among these immunoassays, the so-called “immunochromatographic” devices which particularly lend to certain analytes are known.
The immunochromatography consists in a solid phase diagnostic method, using dry chemistry and lateral migration on an inert membrane.
This kind of immunoassay implements a porous support in a strip form, in which and/or on which are inserted in a dry form the reagents required for performing the test.
The porous support is treated so that a recognition reaction between specific binding partners (generally antigen/antibody) occurs at a capture zone and can be revealed at this level.
In practice, when the liquid sample to be analyzed is deposited on the support, the same migrates along the membrane by a capillary action. The desired analyte is generally captured by a specific capture reagent immobilized on a given zone of the membrane.
The reaction is revealed by a labeled specific detection reagent according to the sandwich method principle.
It is also possible to use a competition type reaction. Generally in this case is used a detection reagent consisting in a labeled analyte which is competitor of the desired analyte for the specific binding with the immobilized capture reagent.
These immunochromatographic devices are generally suitable for a single and domestic use. Indeed, they are of easy and rapid use, requiring only minimal handling since all of the reagents are inserted or comprised in the device.
However, in some particular situations, these immunochromatographic devices are capable of providing results that are not fully reliable.
On one hand, when determining an analyte by a “sandwich” format test, a “hook effect” is observed for certain analytes.
The hook effect is an unwanted effect well known in immunological tests. It occurs when the analyte is present in the sample at a very high concentration.
The hook effect can then result in false negatives, leading to the aberrant conclusion that there is no analyte or it is present at a low concentration in the sample.
Analytes having a hook effect in sandwich type immunoassays generate signal/concentration curves of the Gauss curve type.
For a given signal, two concentrations are then possible, one being low and the other high, for the desired analyte when reading the result at a defined time.
A competition test allows for two different signals to be obtained for two concentrations respectively different from the analyte to be dosed.
But very rapidly, the competition tests also show their limitations, since there is signal extinction at relatively low concentrations of the analyte of interest.
A solution commonly adopted to overcome the drawbacks of these sandwich and competition tests consists in dosing the analyte from a dilution range of the liquid sample.
But the use of such a dilution range is not suitable for domestic use. Furthermore, the use of a dilution range of the sample requires additional handlings and an increased consumption of single use test devices because each sample is tested at the different dilutions.
A solution for that purpose is for example described in WO 2007/023372, relating to a device for determining an analyte in a liquid sample, comprising a capillary diffusion means on which are materialized:
a) a sample depositing or receiving zone;
b) an upstream release zone comprising an analyte-specific detection reagent conjugated to a visible and/or measurable marker, which is free to migrate by capillary diffusion in a wet state in the capillary diffusion means; and
c) two downstream capture zones comprising, successively in the capillary diffusion direction, on one hand, an analyte-specific capture reagent and, on the other hand, the analyte or an analogue of the analyte, which is immobilized.
The detection reagent and the capture reagent, specific of the analyte, allow for the determination of the analyte in the liquid sample by a sandwich test; then the same analyte-specific detection reagent and the analyte, or the analyte analogue, immobilized, allow for the determination of the analyte of interest in the liquid sample by a competition test.
In such a device, the detection reagent is deposited in excess at a single release zone which is formed upstream of the complementary capture zones.
Despite of its interest, the inventors found that this excess of detection reagent upstream of the capture zones is not satisfactory, in that non specific reactions, a strong background and migration problems are frequently observed.
On the other hand, certain immunochromatographic devices are structured for the simultaneous determination of several analytes.
Such an immunochromatographic device, for example, described in EP-1 657 550, comprises advantageously a capillary diffusion means on which are materialized:
a) a sample depositing or receiving zone;
b) an upstream release zone comprising a mixture of detection reagents, each specific of one of the analytes, conjugated to a visible and/or measurable marker, which are free to migrate by capillary diffusion in a wet state in the capillary diffusion means; and
c) downstream capture zones each comprising a capture reagent which is specific of one of the analytes, said downstream capture zones being distributed successively according to the direction of migration.
Again, the detection reagents are deposited at the single release zone which is materialized upstream of the capture zones, taking into account the capillary migration direction.
However, the inventors found again that this mixture of detection reagents, upstream of the different capture zones, is not satisfactory because of cross reactions between the different detection reagents and also mutual specific activity inhibition between reagents.
Immunochromatographic devices comprising a release zone of the detection reagent(s), which is formed upstream of a plurality of capture zones, are not thus satisfactory.
There is therefore a need for new capillary diffusion determination devices both for the determination of a single analyte and for the determination of several analytes, allowing to alleviate the problems generated especially by the excess and/or the mixture of detection reagent(s) reported upstream of a plurality of successive capture zones.